The Nuclear Receptor REV-Erbα Modulates Th17 Cell-Mediated Autoimmune Disease

The nuclear receptor REV-ERBα modulates Th17 cell-mediated autoimmune disease

Christina Changa,b, Chin-San Looa,b, Xuan Zhaoc, Laura A. Soltd, Yuqiong Lianga, Sagar P. Bapata,c,e, Han Choc, Theodore M. Kameneckad, Mathias Leblanca, Annette R. Atkinsc, Ruth T. Yuc, Michael Downesc, Thomas P. Burrisf, Ronald M. Evansc,g,1, and Ye Zhenga,1

aNOMIS Center for Immunobiology and Microbial Pathogenesis, The Salk Institute for Biological Studies, La Jolla, CA 92037; bDivision of Biological Sciences, University of California San Diego, La Jolla, CA 92093; cGene Expression Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037; dDepartment of Molecular Therapeutics, The Scripps Research Institute, Jupiter, FL 33458; eMedical Scientist Training Program, University of California San Diego, La Jolla, CA 92093; fCenter for Clinical Pharmacology, Washington University School of Medicine and St. Louis College of Pharmacy, St. Louis, MO 63104; and gHoward Hughes Medical Institute, The Salk Institute for Biological Studies, La Jolla, CA 92037

Contributed by Ronald M. Evans, July 29, 2019 (sent for review May 9, 2019; reviewed by Ming O. Li and David D. Moore) T helper 17 (Th17) cells produce interleukin-17 (IL-17) cytokines In this study, we show that REV-ERBα is also a key feedback and drive inflammatory responses in autoimmune diseases such as regulator of RORγt in Th17 cells. REV-ERBα is specifically up- multiple sclerosis. The differentiation of Th17 cells is dependent on regulated during Th17 differentiation and plays a dual role in the retinoic acid receptor-related orphan nuclear receptor RORγt. Th17 cells. When expressed at a low level, REV-ERBα promotes Here, we identify REV-ERBα (encoded by Nr1d1), a member of the RORγt expression via the suppression of negative regulator nuclear hormone receptor family, as a transcriptional repressor NFIL3 as reported previously (15, 16). At high expression level, that antagonizes RORγt function in Th17 cells. REV-ERBα binds to REV-ERBα directly competes with RORγt binding to the loci of ROR response elements (RORE) in Th17 cells and inhibits the Th17 signature genes and suppresses Th17 effector function. Ele- expression of RORγt-dependent genes including Il17a and Il17f. vated REV-ERBα activity also ameliorates Th17-driven autoim- Furthermore, elevated REV-ERBα expression or treatment with a mune disease experimental autoimmune encephalomyelitis (EAE). synthetic REV-ERB agonist significantly delays the onset and impedes Our results suggest that modulating REV-ERBα activity could the progression of experimental autoimmune encephalomyelitis provide a way to manipulate Th17 cells in autoimmune diseases. INFLAMMATION

(EAE). These results suggest that modulating REV-ERBα activity may IMMUNOLOGY AND be used to manipulate Th17 cells in autoimmune diseases. Results REV-ERBα Is Highly Expressed during Th17 Cell Differentiation. In an transcriptional repressor | autoimmunity | nuclear receptors effort to identify novel players in the nuclear hormone receptor superfamily that are involved in T cell function, we conducted ex- helper 17 (Th17) cells are the drivers of inflammatory repression profiling of nuclear hormone receptors in different T Tsponses in a large number of autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, and psoriasis (1, 2). The Significance orphan nuclear receptor RORγt is the lineage-specific transcription factor that regulates the differentiation of Th17 cells Th17 cells are a subset of T cells that produce interleukin 17 (3). RORγt expression is induced specifically under Th17 dif- and other proinflammatory cytokines. They are involved in the ferentiation condition. Once expressed, RORγt in turn binds to development of autoimmune diseases such as multiple sclero- the loci of Th17 signature genes Il17a and Il17f and up-regulates sis and rheumatoid arthritis. Previous studies illustrated that γ their expression (4). Several small-molecule RORγt antagonists ROR t is a driver for Th17 cell differentiation. Here, we reveal α γ α γ were identified that can inhibit Th17 cell differentiation and REV-ERB as an antagonist of ROR t. REV-ERB and ROR t effector function (5–8). These findings suggested that RORγt share the same DNA binding motif. REV-ERB can inhibit the expression of RORγt target genes and suppress RORγt-driven inhibitors could be developed for treatment of autoimmune Th17 cell differentiation. Treatment with a synthetic REV-ERB diseases. However, RORγt is also known for its critical role in + + agonist delays the onset and impedes the progression of ex- promoting survival of CD4 CD8 double-positive (DP) thymo- perimental autoimmune encephalomyelitis (EAE), a mouse γ cytes. A recent study showed that ROR t inhibitor treatment model of multiple sclerosis. Taken together, our study suggests leads to not only reduced DP thymocyte numbers but also lim- that modulating REV-ERBα activity may be used to manipulate ited T cell repertoire diversity (9). Therefore, it is still a chal- Th17 cells in autoimmune diseases. lenge to develop a safe strategy to inhibit RORγt activity in Th17 cells in vivo. Author contributions: C.C., M.D., R.M.E., and Y.Z. designed research; C.C., C.-S.L., X.Z., Beyond their critical roles in Th17 cell differentiation, members L.A.S., Y.L., S.P.B., H.C., and T.M.K. performed research; C.C., C.-S.L., L.A.S., M.L., A.R.A., M.D.,T.P.B.,R.M.E.,andY.Z.analyzeddata;andC.C.,R.T.Y.,M.D.,andY.Z.wrote of the ROR family are known to be key players in the circadian the paper. regulatory machinery, where they function as transcriptional ac- Reviewers: M.O.L., Memorial Sloan Kettering Cancer Center; and D.D.M., Baylor College tivators to turn on the expression of circadian genes (10, 11). In of Medicine. the circadian system, RORs’ transcriptional activity is opposed by The authors declare no conflict of interest. a pair of repressors, REV-ERBα and REV-ERBβ.LikeRORs, This open access article is distributed under Creative Commons Attribution-NonCommercial- REV-ERBs are also members of the nuclear hormone receptor NoDerivatives License 4.0 (CC BY-NC-ND). family and play critical roles in circadian and metabolic regula- Data deposition: RNA-Seq data reported in this paper have been deposited in the Na- tions (12). REV-ERBs recognize the same RORE DNA sequence tional Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) database (accession no. PRJNA293472). ChIP-Seq data have been deposited in Gene Expression as RORs and function as transcriptional repressors to suppress the Omnibus (GEO), www.ncbi.nlm.nih.gov/geo (accession no. GSE72271). expression of ROR target genes (13, 14). Although the antagonistic 1To whom correspondence may be addressed. Email: [email protected] or [email protected]. relationship between ROR and REV-ERB was well established in This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the circadian rhythm system, it is not clear if a similar interaction 1073/pnas.1907563116/-/DCSupplemental. exists in the T cell lineage.

www.pnas.org/cgi/doi/10.1073/pnas.1907563116 PNAS Latest Articles | 1of9 Downloaded by guest on September 30, 2021 REV-ERBα AB1.0 0.8 RORγt Tbet Foxp3 Gata3 Th1 0.8 Th17 5 0.8 4 0.6 iTreg 4 0.6 0.6 3 0.4 3 0.4 0.4 2 0.2 2 0.2 0.2 1 1 Relative expression 0.0 Day(s) 0.0 Day(s) 0 0.0 0 012 3 4 012 3 4 g 7 g re ve Th0 Th1Th2 iTre Th0 Th1Th2 Th0 Th1Th2 Naive Th17 Naive Th1 iT Nai Th17iTreg C Th1 Th17 Days RORγt REV-ERBα REV-ERBβ 12 34 1234 2.5 1.5 0.6 REV-ERBα Relative expression 2.0 1.0 0.4 REV-ERBβ 1.5 1.0 RORγ 0.5 0.2 0.5 Tbet 0.0 0.0 0.0 7 ve ve Th0 Th1 Th0 Th1Th2 Th0 Th1Th2 Th2Th17iTreg β-Actin Naive Th1 iTreg Nai Th17iTreg Nai

100 D hRORγt hTbet hREV-ERBα E IL-17A MIGR1 REV-ERBα REV-ERBβ 80 γ+ 15 60 3 *** 60 P=0.06 40 Donor 10 40 2 0 0 0 20 %IFN #1 Th1 0 5 20 **** 1 99.8 99.7 99.8 *** 50 0 **** 0 0 40 20 60 8 * 30 *** 41.3 10.5 26.7 15 6 20 40

Relative expression ** Donor 10 4 Th17 10 % IL-17A+ 20 0 #2 5 2 α β 0 0 0 0.26 1.71 0.213 Th1 Th17 Th1 Th17 Th1 Th17 Control IFNγ

REV-ERBREV-ERB

#2 #1

FGl

#2-ERBαREV

#1-ERBβREV #2

#1 #1-ERBαREV

βREV

BαREV

BβREV ERB

KEGG Pathway Term Count P value ERBα

-ERB

-ERBβREV

-ER

- -ERBβ

-ER

-ERBα - -ERBα #1 27 7.38E-07

T cell receptor signaling pathway MigR1 Control #1

MigR1 Control #2

REV

MigR1 Control #2 MigR1 Control #1

MigR1 Contro MigR1 Control

Jak-STAT signaling pathway 28 3.29E-05 Il-17a Pim1 Vav3 Il-17f Spry1 Ccl3 Chemokine signaling pathway 26 0.00334 Il-23r Stat2 Ncf1 5 0.01452 Tgfb3 Stat1 Ccr5 Circadian rhythm signaling Csf2 Il12rb Chemokine Cc14 27 Cytokine-cytokine receptor 0.05854 Ccl20 Il7r interaction Cysltr1 Stat5a Cd40 Ltb4r1 Stat3 Inhba Furin Irf9 Il9 Fam124b Jak2 Fas Vax2 Akt2 Il1r2

Th17 differentiation and function Pik3ca Kit

Jak-Stat pathway Tnfsf11 Cry1 Akt3 interaction Arnt1 Myc Il15ra Cd27 Npas2 Il2ra Tnfsf8 Cytokine-cytokine receptor rhythm Bhlhe40 Socs2 Circadian Il2rb Il12rb2 Pik3cg Stat4

log2 fold change

Fig. 1. REV-ERBα is up-regulated in Th17 cells and inhibits the expression of Th17 signature genes. (A) mRNA expression of REV-ERBα, REV-ERBβ, as well as T cell lineage specifying transcription factors T-bet, Gata3, RORγt, and Foxp3, in naïve T cells and Th0, Th1, Th2, Th17, and iTreg cells differentiated for 3 d in vitro. (B) REV-ERBα and RORγt mRNA expression in Th1, Th17, and iTreg cells over 4 d of in vitro differentiation. (C) Protein expression of REV-ERBα, REV- ERBβ, RORγt, and T-bet during in vitro differentiation of Th1 and Th17 cells over 4 d. (D) mRNA expression of RORγt, T-bet, and REV-ERBα in human CD4+ T cells activated under Th1 and Th17 polarizing conditions for 6 d. (E) FACS analysis of IL-17A and IFN-γ expression in mouse CD4+ T cells activated under Th1 and Th17 polarizing conditions and transduced with MIGR1, REV-ERBα or REV-ERBβ retroviral vectors. Data are representative of 3 independent experiments with triplicate wells for each condition. (F) KEGG pathway analysis of genes differentially expressed in REV-ERBα and MIGR1 retrovirally transduced Th17 cells. (G) Heat map of functional groups of differentially expressed genes in Th17 cells transduced with MIGR1, REV-ERBα, or REV-ERBβ retroviral vectors. Relative fold change was normalized to the average of each row in the matrix. Data represents mean ± SEM. Statistical analyses were performed using unpaired 2-tailed Student’s t test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

2of9 | www.pnas.org/cgi/doi/10.1073/pnas.1907563116 Chang et al. Downloaded by guest on September 30, 2021 Il17a RORE 10% input ABIl17a promoter+CNS5 * 400

*** t-HA t-HA α-Flag α-Flag 300 RVB RORγ Control RVB RORγ Control

200

RFU Anti-Flag

100 Anti-HA 0 t ol γ r 1) RVBα (1: (1:0.5) Cont ROR α (1:0.25) D De novo

t+RVBγt+RVBα t+RVBα Annotated RORγt OR ORγ RORγR R

CG10kb Il17a Cry1 Gmpr REV-ERBα ** * *** ** RORγt 1.0 1.0 1.0 Il17a

0.5 0.5 0.5 10kb %ofinput INFLAMMATION

0.0 0.0 0.0 REV-ERBα IMMUNOLOGY AND α t α α γ γt t IgG IgG IgG RB ROR ROR RORγ RORγt REV-ERB REV-ERB REV-E Il17f

E RORγt H REV-ERBα ChIP RORγt ChIP REV-ERBα 2.0 0.8 MigR1 Il-17a 1.5 0.6 REV-ERBα 15211 Il-17f IL-23r 1.0 0.4 2630 Tgfb3 0.5 0.2

6254 % of input

0.0 0.0

a a Il17 Gmpr Il17 Gmpr

FIREV-ERBα ChIP RORγt ChIP KEGG Pathway Term Count P Value 1.5 2.0 T cell receptor signaling pathway 32 4.15E-08 1.5 MigR1 Cytokine-cytokine receptor 50 7.76E-08 1.0 RORγt interaction 1.0 Jak-STAT signaling pathway 34 1.87E-06 0.5 MAPK signaling pathway 47 1.35E-05 % of input 0.5 Wnt signaling pathway 27 9.61E-04 0.0 0.0 Chemokine signaling pathway 31 1.06E-03

Il17a Il17a Gmpr Gmpr

Fig. 2. REV-ERBα directly competes with RORγt and represses Th17 signature gene expression. (A) Luciferase assays of EL4 T cells cotransfected with an Il17a luciferase reporter, and combinations of RORγt and REV-ERBα at various ratios, with the amount of RORγt transfected remaining constant. Renilla luciferase activity was used as internal control. (B) Western blot to detect the binding of HA-tagged RORγt and Flag-tagged REV-ERBα expressed in CD4 T cells to biotinylated oligonucleotides containing RORE motif derived from the Il17a CNS5 enhancer. (C) ChIP-qPCR to detect the binding of Il17a CNS5 enhancer, Cry1 (positive control) and Gmpr (negative control) by REV-ERBα and RORγt in Th17 cells. (D–G) Analysis of Th17 cell REV-ERBα ChIP-seq data along with published RORγt ChIP-seq data. (D) Alignment of de novo generated REV-ERBα binding sequence to annotated RORγt binding sequence. (E) Venn diagram depicting the numbers of unique and shared genes bound by REV-ERBα and RORγt. (F) KEGG pathway analysis of REV-ERBα bound genes. (G) Trace analysis of ChIP-seq data visualized on the UCSC genome browser showing overlapping binding sites of REV-ERBα and RORγtatIl17a and Il17f loci. (H and I) ChIP-qPCR to detect changes in REV-ERBα and RORγt binding to the Il17a locus in response to ectopic expression of REV-ERBα (H) or RORγt(I). Data represents mean ± SEM. Statistical analyses were performed using unpaired 2-tailed Student’s t test (*P < 0.05, **P < 0.01, ***P < 0.001).

Chang et al. PNAS Latest Articles | 3of9 Downloaded by guest on September 30, 2021 helper cell subsets. We noticed that, similar to RORγt, REV-ERBα ERBα and RORγt. As shown in Fig. 2B, both REV-ERBα and expression was uniquely up-regulated in Th17 cells at both mRNA RORγt bind to the RORE motif. To determine if the REV- and protein levels (Fig. 1 A–C). The differences in the kinetics of ERBα:CNS5 interaction occurs in vivo, we performed chromatin REV-ERBα mRNA and protein expression are likely due to a immunoprecipitation (ChIP) experiments in Th17 cells with anti- tightly regulated protein degradation pathway of REV-ERBα (17, REV-ERBα and anti-RORγt antibodies. Indeed, both REV- 18). Furthermore, REV-ERBα expression was significantly higher ERBα and RORγt bound to the CNS5 region in Th17 cells in human Th17 cells relative to Th1 cells (Fig. 1D). The unique (Fig. 2C). These findings suggest that REV-ERBα can directly expression pattern of REV-ERBα suggested that it may play a role repress Il17a expression by binding to the Il17a CNS5 enhancer. in the regulation of Th17 cells. Previous studies on circadian reg- To identify genome-wide REV-ERBα target genes in Th17 ulation demonstrated that, by binding to the same RORE motifs, cells, we performed REV-ERBα ChIP-seq assays (23). As RORs activate transcription of their target genes, whereas REV- expected, the de novo REV-ERBα binding motif is highly similar ERBs act as repressors of the same targets (13, 14). We hypothe- to the established RORγt binding motif (Fig. 2D). When com- sized that REV-ERBs may suppress Th17 cell differentiation and pared to previously published RORγt ChIP-seq data, about 30% function by antagonizing RORγt. of the 8,884 REV-ERBα binding sites in Th17 cells are also RORγt binding sites (Fig. 2E) (4). It is expected that a large Ectopic Expression of REV-ERBα Inhibits the Expression of Th17 proportion of REV-ERBα binding peaks are different from Signature Genes. To assess the role of REV-ERBs in Th17 RORγt binding peaks despite their shared binding motif. This is cells, we examined the effects of ectopic expression of REV- partly due to the finding that REV-ERBα can target DNA in- ERBs on Th1 and Th17 cell differentiation. Retroviral expres- directly by interacting with other transcription factors (24). sion of REV-ERBα during Th17 differentiation significantly KEGG pathway analysis of genes bound by both REV-ERBα suppressed interleukin-17A (IL-17A) production compared to and RORγt revealed that they are enriched with genes in- T cells transduced with control vector MIGR1 (Fig. 1E). The volved in T cell signal and cytokine/chemokine pathways (Fig. inhibitory effect of REV-ERBα is specific to Th17 cells, as it did 2F). In addition to Il17a, several other Th17 cell signature genes, not suppress interferon (IFN)-γ expression in Th1 cells. Ectopic including Il17f, Il23r, and Tgfb3, were also identified as direct expression of REV-ERBβ showed a modest negative impact on targets of REV-ERBα (Fig. 2 E and G and SI Appendix, Fig. S2). Th17 cells (Fig. 1E). Th17 differentiation can also be driven The finding that REV-ERBα binds to a large number of RORγt by ectopic expression of RORγt in T cells cultured without target genes suggests that REV-ERBα inhibits Th17 cell differ- Th17 polarizing cytokines (3). We found that coexpression of entiation through direct suppression of the expression of key REV-ERBα along with RORγt also led to significant decrease Th17 cell signature genes. To further test this hypothesis, we of IL-17A expression (SI Appendix, Fig. S1), suggesting that examined whether ectopic REV-ERBα expression decreases the REV-ERBα can suppress RORγt-dependent IL-17A expres- binding of RORγttoIl17a by ChIP-qPCR. Indeed, RORγt sion. To evaluate the genome-wide effects of REV-ERBs’ ec- binding at the Il17a locus decreased significantly in cells trans- topic expression in Th17 cells, we performed RNA-sequencing duced with a REV-ERBα expressing retroviral vector (Fig. 2H). (RNA-seq) analysis of Th17 cells retrovirally transduced with Conversely, ectopic expression of RORγt diminished REV- REV-ERBα, REV-ERBβ, or MIGR1 control vector (19). KEGG ERBα binding at the Il17a locus (Fig. 2I). pathway analysis of the differentially expressed genes indicated that REV-ERBα regulates genes involved in T cell receptor In Vivo Induction of REV-ERBα Expression in T Cells Suppresses EAE signaling, cytokine/chemokine signaling, as well as circadian Disease Progression. Since high levels of REV-ERBα expression rhythm regulation (Fig. 1F). REV-ERBα–transduced cells dif- are inhibitory to Th17 cell differentiation in vitro, we explored ferentially expressed a number of Th17 cell signature genes whether constitutive REV-ERBα expression in T cells could compared with MIGR1-transduced cells, which include Il17a, ameliorate Th17 cell-mediated autoimmune diseases in vivo. We Il17f, Il23r, Csf2, and Tgfb3. Interestingly, most of these genes tested this hypothesis in EAE, a mouse model for multiple scle- were significantly down-regulated by REV-ERBα (Fig. 1G). rosis, as Th17 cells play a critical role in EAE disease develop- REV-ERBβ expression also suppressed most Th17 signature ment. A tetracycline-inducible REV-ERBα transgenic mouse genes, but its impact was modest compared to REV-ERBα (Fig. (TRE-REV-ERBα/Rosa-M2rtTA) (25) was crossed with a 2D2 1G). Therefore, we decided to focus on the role of REV-ERBα TCR transgenic mouse, which carries a TCR that recognizes the in suppressing Th17 cell differentiation and the expression of MOG (myelin oligodendrocyte glycoprotein) peptide (26), to Th17 signature genes. generate a triple transgenic mouse strain (TTg). T cells from the TTg mice express REV-ERBα constitutively under doxycycline + REV-ERBα Directly Competes with RORγt and Represses Th17 Signature treatmentinvivo(SI Appendix,Fig.S3A). CD4 T cells from the Gene Expression. Since REV-ERBs and RORs both recognize TTg mice were activated in vitro under Th17 conditions for 4 d ROREs, we hypothesized that REV-ERBα could directly interact before being adoptively transferred into WT recipient mice to with the Il17a locus and repress its transcription. RORE motifs induce EAE. Mice were given doxycycline water to induce REV- located in CNS5 (also named CNS2), an enhancer 5 kb upstream ERBα expression in transferred 2D2 T cells or normal water. The of the Il17a locus, are critical for optimal expression of Il17a (20– doxycycline-treated group showed delayed EAE disease onset as 22). Using a reporter driven by the Il17a promoter and CNS5 well as slower disease progression compared to mice that were (20), we measured luciferase activity after transfecting RORγt given normal water (Fig. 3A). Elevated REV-ERBα expression with or without REV-ERBα. Cotransfection of REV-ERBα did not affect homing, proliferation, or survival of the transferred + inhibited RORγt-dependent Il17a reporter activity in a dose- CD4 T cells (SI Appendix,Fig.S3B). However, consistent with dependent manner (Fig. 2A). To investigate whether REV- milder disease progression observed in mice treated with doxycy- ERBα can directly bind to ROREs located at the Il17a locus, cline, the frequency of IL-17A producing 2D2 T cells were sig- we performed an in vitro DNA binding assay. Biotinylated oli- nificantly reduced in the CNS tissues of these mice compared to gonucleotides containing the RORE motif derived from the controls (Fig. 3B). Histopathology analysis showed that doxycy- Il17a CNS5 enhancer were incubated with nuclear extracts from cline treatment significantly reduced inflammation levels with a mouse CD4 T cells transduced with either REV-ERBα− or decreasing trend for demyelination in the spinal cord of these RORγt-expressing retroviral vectors. The DNA:protein com- mice (Fig. 3 C and D). These differences were dependent on plexes were then precipitated with streptavidin beads, and REV-ERBα induction because the same doxycycline treatment of Western blots were performed to detect precipitated REV- mice transferred with WT 2D2 T cells did not delay or ameliorate

4of9 | www.pnas.org/cgi/doi/10.1073/pnas.1907563116 Chang et al. Downloaded by guest on September 30, 2021 AB4 ** Control 0.95 4 3.4 Control 3 Dox 2 3 % IL-17A+ 1 6.22 0 89.4 2 * 10 Dox

0.294 **** 8 1.02 Clinical score

1 6 IL-17A

4 %IFNγ+ 0 2 3.78 05101520 0 94.9 Control Dox Day(s) IFNγ

CDH&E 2.5x Luxol Fast Blue Inﬂammation

4 * 3

2 Control 1 0 Demyelination

4 3

Dox 2

1 INFLAMMATION

0 IMMUNOLOGY AND Bar = 200 m Bar = 80 m Bar = 200 m Control Dox

Fig. 3. In vivo induction of REV-ERBα expression in Th17 cells suppresses EAE disease progression. EAE was induced in C57/BL6 mice by adoptive transfer of in vitro differentiated Rosa-M2rtTAxTRE-REV-ERBax2D2 transgenic Th17 cells. Recipient mice were treated with or without Doxycycline water (n = 7 per group) starting 2 d before Th17 cell adoptive transfer and were monitored for EAE disease progression. Mice were analyzed on day 24 after transfer. (A) + Clinical scores of mice induced with EAE. (B) FACS analysis of IL-17A and IFN-γ production of transferred 2D2 CD4 T cells infiltrating in the CNS tissues. (C) Representative H&E and Luxol Fast Blue staining of the spinal cords to show the sites of immune cell infiltration (filled arrow) and demyelination (open arrow). Data represents mean ± SEM. Statistical analyses were performed using 2-way analysis of variance (ANOVA) for EAE clinical score analysis and 2-tailed unpaired Student’s t test for other analysis, comparing the indicated groups (*P < 0.05, **P < 0.01).

EAE disease progression (SI Appendix, Fig. S3C). Thus, elevated Th17 signature genes such as Il17a and Il17f, also resulting in the expression of REV-ERBα in T cells can attenuate Th17 cell- suppression of Th17 cell differentiation. It is worth noting that mediated EAE. overexpression of REV-ERBα exerts a much stronger inhibitory effect on Th17 cells than absence of REV-ERBα expression REV-ERBα Deficiency Impairs Th17 Cell Differentiation. Despite the (Figs. 1E and 4A). suppressive role of REV-ERBα on the expression of Th17 signature genes, a previous study by Hooper and coworkers (15) A Synthetic Agonist of REV-ERBα Can Inhibit Th17 Cell Differentiation showed that REV-ERBα–deficient T cells were also defective in In Vitro and Ameliorate EAE In Vivo. Structural studies have shown Th17 differentiation. It was proposed that in the absence of that REV-ERBs contain a ligand binding domain, and their REV-ERBα, expression of NFIL3 increases, which, in turn, activity can be modulated by specific ligands (27). Heme, the suppresses Th17 cell development by directly binding to the prosthetic group in hemoglobin, was identified as an endogenous RORγt promoter and repressing its expression. We assessed the agonist that binds to REV-ERBs and potentiates their activity effect of REV-ERB ablation in Th17 cell differentiation and (28, 29). Efforts have also been made to generate synthetic REV- function. The REV-ERBα/β knockout T cells showed a moder- ERB agonists with higher specificity and fewer side effects. Two ate reduction in Th17 differentiation measured by IL-17A ex- chemical compounds, SR9009 and SR9011, bind specifically to pression (Fig. 4A), consistent with the findings by Hooper’s REV-ERBs and modulate their activity, and exhibited favorable group. To test the impact of REV-ERB deletion in vivo, we pharmacokinetic properties when tested in mice (30–32). Since utilized T cell-specific REV-ERBα/β conditional knockout mice increased REV-ERBα expression suppresses Th17 cell differ- (REV-ERBαfl/fl/βfl/fl CD4Cre) (14). The REV-ERB conditional entiation and function, we tested if potentiating REV-ERB ac- knockout mice and WT controls were immunized with MOG/ tivity via agonist treatment could have a similar effect on Th17 CFA to induce EAE. Disease development was monitored for 3 cells. First, we cultured naïve mouse CD4 T cells under Th1, Th17, wk and followed by analysis of T cell composition in CNS tissues. or iTreg differentiation conditions with or without SR9009. Mice carrying REV-ERB null T cells developed milder EAE SR9009 treatment significantly inhibited Th17 cell differentiation accompanied with reduced IL-17A–producing T cells in the CNS but did not affect Th1 or iTreg differentiation (Fig. 5 A and B). tissues (Fig. 4 B and C). These results suggest that REV-ERBα Similarly, Th17, but not Th1, differentiation of human CD4 T cells expression needs to be tightly regulated for robust Th17 cell was significantly inhibited by SR9009 treatment (Fig. 5C). To test differentiation. Insufficient REV-ERBα activity leads to de- the specificity of SR9009, we cultured CD4 T cells isolated from creased Th17 cell differentiation due to increased levels of REV-ERBα/β conditional knockout and WT control mice under NFIL3, which suppress RORγt expression, whereas at high levels Th17 differentiation condition with or without SR9009 and mea- REV-ERBα outcompetes RORγt for regulatory binding sites in sured their IL-17A expression. While Th17 differentiation of WT

Chang et al. PNAS Latest Articles | 5of9 Downloaded by guest on September 30, 2021 A RVBαfl/fl/βfl/fl control RVBαfl/fl/βfl/fl CD4Cre maintained their remitting state, whereas vehicle-treated control 25 mice developed additional episodes of EAE symptoms (Fig. 5I). 20 * Furthermore, the frequency of IL-17A–producing CD4 T cells was 15 also significantly reduced in the CNS tissues of SR9009-treated 19.7% 14.6% 10

%IL-17A+ 5 mice compared to controls (Fig. 5J). These results demonstrate 0 that modulating in vivo REV-ERB activity by its agonist SR9009 effectively suppresses development and progression of Th17 cell- control fl/fl CD4Cre mediated EAE. IL-17A fl/fl /fl /β fl /fl /β α fl α RVB Discussion RVB In this study, we demonstrated a role for REV-ERBα in the BCRVBαfl/fl/βfl/fl control fl/fl fl/fl 4 RVBα /β CD4Cre 40 40 regulation of Th17 cell differentiation and function in addition to

30 its established roles in circadian rhythm and metabolism. REV- 30 γ 3 * ERBα is induced during Th17 cell differentiation and directly 20 20 γ %IFN- competes with ROR t by binding to the RORE sites to repress the 2 % IL-17A 10 10 **** expression of key Th17 cell signature genes such as Il17a and Il17f. 0 0 1 At the same time, normal RORγt induction is also dependent on EAE disease score e α control control repression of Nfil3 by REV-ERB (15). This is substantiated by l/fl fl/fl fl CD4Cre f fl CD4Cr 0 fl/ fl/ fl β fl β / / fl/ / reduction of IL-17A production in vitro and milder EAE pheno- 0 2 4 6 8 9 fl fl /β /fl /β 12 14 16 18 20 22 α fl/ α fl B α B α V type in vivo as a result of T cell-specific REV-ERB ablation. These Days RV R RVB RVB observations suggest that REV-ERBα serves as a feedback regu- γ Fig. 4. REV-ERBα deficiency impairs Th17 cell differentiation. (A) CD4+ lator for ROR t in T cells, and its expression needs to stay at the T cells from REV-ERBαfl/fl/βfl/fl control and CD4Cre REV-ERBαfl/fl/βfl/fl mice were right level for optimal Th17 differentiation. activated under Th17 polarizing condition. After 3 d of culturing, IL-17A A recent study by Amir et al. reported similar results showing production was analyzed by flow cytometry. FACS plots shown were rep- reduced Th17 activity when REV-ERB expression is increased resentative of 3 independent experiments. (B and C) REV-ERBαfl/fl/βfl/fl con- (33). However, the same study also showed Th17 differentiation trol and CD4Cre REV-ERBαfl/fl/βfl/fl mice were immunized with MOG/CFA to was enhanced in REV-ERBα knockout T cells, which differs induce EAE. (B) EAE disease progression scores. (C) IL-17A and IFN-γ pro- from our results and the study performed by Yu et al. (15). One duction in the CNS infiltrating CD4 T cell population at endpoint. Data primary difference between the 2 studies is that T cell-specific represents mean ± SEM. Statistical analyses were performed using unpaired ’ < REV-ERB conditional knockout mice were used in our study, 2-tailed Student s t test or 2-way ANOVA for EAE disease scores (*P 0.05, α ****P < 0.0001). while REV-ERB germline knockout mice were used in the study by Amir et al. Since REV-ERBα germline knockout mice carried severe defects in circadian and metabolic regulation, it is possible T cells was suppressed by SR9009 treatment, IL-17A expression in that these perturbations originated outside of the immune system REV-ERB null T cells was not affected (Fig. 5 D and E). These rendered T cells more inflammatory under Th17-inducing condi- results suggest that SR9009 can inhibit Th17 differentiation by tions. Additionally, differences in gut microbiota between mouse modulating REV-ERB activity. facilities might also contribute to the contradictory results. α To investigate the molecular mechanism of SR9009’s effect in Given the key role REV-ERB plays in Th17 cells, we ex- α Th17 cells, we examined the recruitment of NCoR, a corepressor plored if tuning REV-ERB activity can influence Th17 differ- that binds to REV-ERB and represses its target gene expression entiation and function. Our results showed that elevated REV- α (24). ChIP-qPCR assay showed that NCoR recruitment to the ERB expression in T cells or treatment with REV-ERB ligand SR9009 suppresses Th17 cell differentiation in vitro and inhibits Il17a CNS5 enhancer increased significantly in the presence of α SR9009, indicating that SR9009 can target the IL17A pathway the development of EAE in vivo. Although specific REV-ERB directly via enhancing NCoR:REV-ERB interaction at the Il17a induction in T cells is sufficient to ameliorate EAE, SR9009 treatment in mice might also impact non-Th17 cells. A previous locus (Fig. 5F). α The inhibitory effect of SR9009 on Th17 differentiation in vitro study demonstrated that REV-ERB could suppress macro- phage expression of IL-6, a key cytokine for Th17 cell differen- compelled us to test if it could also exert similar modulating effect tiation (34). We also observed that subsets of gamma/delta on Th17-induced autoimmune diseases in vivo. Unlike RORγt T cells and regulatory T cells could express high levels of REV- antagonists, SR9009 treatment did not skew T cell development in ERBα, although the significance of these cell subsets in EAE the thymus or grossly affect T cell activation state in the periphery pathogenesis is currently unclear and requires further charac- (SI Appendix,Fig.S4). terization. A recent study raised concern on the specificity of To test the effect of SR9009 treatment in mouse EAE models, SR9009 by demonstrating that SR9009 could exert REV-ERB we immunized C57/BL6 mice with MOG/CFA and started in- independent effects in certain tissues, such as mouse embryonic jection of SR9009 or vehicle control 7 d after initial immunization. stem cells and hepatocytes (35). In our experiments, SR9009 Mice treated with SR9009 showed significantly delayed onset and treatment only affects Th17 differentiation in WT T cells, not slower progression of EAE compared to vehicle-treated control REV-ERBα/β double knockout T cells (Fig. 5 D and E), sug- group (Fig. 5G). SR9009 treatment also reduced the frequency of gesting that SR9009s inhibitory effects on Th17 cells is REV- – IL-17A producing CD4 T cells infiltrating the CNS tissues during ERB dependent. EAE (Fig. 5H). We next explored the efficacy of REV-ERB ag- A concerted effort has been made to identify RORα/γ antagonist treatment on mice that have already developed EAE. We onists for treatment of Th17-related autoimmune diseases (5–8). induced EAE in SJL mice, which upon PLP (myelin proteolipid In fact, a recent clinical trial on an RORγ antagonist showed protein) peptide immunization, exhibit disease progression in re- encouraging results in psoriasis patients (36). In addition to Th17 mitting and relapsing patterns mimicking the development of cells, RORγt is also highly expressed in developing T cells in the multiple sclerosis in humans. SR9009 treatment of SJL mice thymus. A recent report showed that RORγ antagonist treat- during the primary phase of EAE showed inhibitory effects similar ment leads to DP thymocyte apoptosis and reshapes the T cell to its effects in C57/BL6 mice (data not shown). When SR9009 repertoire by skewing TCRα rearrangement (9). Although lim- was administered in the remitting phase, SR9009-treated mice iting the diversity of the T cell repertoire could be beneficial

6of9 | www.pnas.org/cgi/doi/10.1073/pnas.1907563116 Chang et al. Downloaded by guest on September 30, 2021 ABDMSO SR9009 50 Th1 50 Th17 30 iTreg **** 38.9 37.5 40 40

Empty + Th1 γ 30 30 20 20 20

%IFN 10 %Foxp3+ 10 %IL-17A+ 10 IFNγ 0 0 0

36.9 27.8 Th17 Foxp3 DMSO DMSO DMSO SR9009 SR9009 SR9009

C hTh1 hTh17 20 20 * IL-17A 15 15

5 10 10 17.3 18.7 %IFNγ+ 4 5 IL-17A % 5 iTreg Foxp3 3 0 0

0 DMSO DMSO SR9009 SR9009 IL-17A D DMSO SR9009 EF DMSO NCoR ChIP 30 SR9009 DMSO 26.1 18 ﬂ/ﬂ fl/fl **** RVBα /β 0.025 ** SR9009 control 20 0.020 0.015 INFLAMMATION

10 0.010 IMMUNOLOGY AND %IL-17A+

%ofinput 0.005 Foxp3 0 20.6 21 l fl 0.000 /f fl/fl fl/fl fl fl/ RVBα /β a /β /β 7 CD4Cre fl/fl fl/fl Il1 Gmpr

control RVBα RVBαCD4Cre IL-17A GI4 4

Vehicle Vehicle 3 SR9009 3 SR9009 treatment start 2 2 treatment start Clinical Score Clinical Score **** 1 1 ****

0 0 0 5 10 15 0 5 10 15 20 25 30 Day(s) Days IFNγ H IL-17A J IL-17A IFNγ * 25 * 20 30 30 *** 20 15 20 20 15 10 10 10 10 %IFNγ+ %IFNγ+ 5 5 %IL-17A+ %IL-17A+ 0 0 0 0

Vehicle Vehicle Vehicle VehicleSR9009 SR9009 SR9009 SR9009

+ Fig. 5. REV-ERB agonist SR9009 inhibits Th17 differentiation and suppresses EAE. (A and B) Mouse CD4 T cells were activated under Th1, Th17, and iTreg polarizing conditions and treated with DMSO or SR9009. IFN-γ, IL-17A, and Foxp3 expression in Th1, Th17, and iTreg cells, respectively, were analyzed by flow + cytometry (n = 3). (C) IFN-γ and IL-17A production in human Th1 and Th17 polarized cells treated with either DMSO or SR9009. (D and E) CD4 T cells from REV-ERBαfl/fl/βfl/fl control and CD4Cre REV-ERBαfl/fl/βfl/fl mice were activated under Th17 polarizing condition and treated with DMSO or SR9009. IL-17A expression in Th17 cells was analyzed by flow cytometry (n = 5). (F) ChIP-qPCR to detect enhanced NCoR recruitment to the Il17a locus in response to SR9009. (G) EAE disease progression of C57/BL6 mice that were immunized with MOG/CFA and treated with vehicle control or SR9009 via i.p. injections starting on day7 after immunization (n = 5–6 per group). (H) IL-17A and IFN-γ production in the CNS infiltrating CD4 T cell population at endpoint. (I) EAE disease progression of SJL mice immunized with PLP/CFA and treated with vehicle control or SR9009 daily starting at the beginning of the relapsing phase of EAE as indicated by the arrow (n = 9 to 10 per group). (J) IL-17A and IFN-γ production in the CNS infiltrating CD4 T cell population at endpoint. Data represents mean ± SEM. Statistical analyses were performed using 2-way ANOVA for EAE clinical score analysis and 2-tailed unpaired Student’s t test for other analysis, comparing the indicated groups (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Chang et al. PNAS Latest Articles | 7of9 Downloaded by guest on September 30, 2021 in some autoimmune disease settings, its long-term effect could reference genome using the Bowtie2 aligner with standard parameters also increase the risk to cancer and certain infections. In contrast that allow up to 2 mismatches in the read. Peak calling, motif analyses, and to RORγt, the low expression levels of REV-ERBα and REV- other data analysis were performed using HOMER, a software suite for ChIP- ERBβ in thymocytes and our own results (SI Appendix, Fig. S4) seq analysis as described previously (14). Visualization of ChIP-Seq results suggest that REV-ERB agonists will not likely have the same im- was achieved by uploading custom tracks onto the University of California, pact on thymocytes and the T cell repertoire as RORγ antagonists Santa Cruz (UCSC) genome browser. ChIP-seq data can be accessed in the (37). Therefore, a strategy of targeting REV-ERB alone or in National Center for Biotechnology Information (NCBI) GEO database under the accession no. GSE72271. combination with RORγ may provide a unique advantage in developing treatments for Th17 cell-mediated autoimmune diseases. RNA-Seq and Data Analysis. RNA-seq libraries were prepared from 100 ng of Materials and Methods total RNA (TrueSeq v2, Illumina) and single-ended sequencing was performed on the Illumina HiSeq 2500. Read alignment and junction finding was ac- α Mice. Rosa-M2rtTA, TRE-REV-ERB , and 2D2 transgenic mice were purchased complished using STAR (39) and differential gene expression with Cuffdiff 2 from Jackson Laboratory. The 3 transgenic lines were crossed to generate (40). Student’s t test was performed to generate a list of differentially Rosa-M2rtTAxTRE-RVBx2D2 triple transgenic mice. REV-ERBαfl/fl/βfl/fl mice expressed genes (P < 0.05), which was then run through KEGG pathway were generated previously (14). CD4Cre transgenic, C57BL/6, SJL/J, and + analysis on DAVID (41, 42) to examine enriched functional groups. Heatmaps Ly5.1 congenic mice were purchased from the Jackson Laboratory. All mice were generated on Matrix2png (43). RNA-seq data can be accessed in the were maintained in the Salk Institute specific pathogen free (SPF) animal facility in accordance with the protocols approved by the Institutional NCBI Sequence Read Archive under accession no. SRP062715. Animal Care and Use Committee (IACUC) at the Salk Institute. EAE Models. For active EAE, mice were immunized s.c. with 200 ng of MOG – – Reverse Transcription and Quantitative PCR. Total RNA was isolated from CD4 (35 55) peptide (BL6 mice) or PLP (139 151) peptide (SJL mice) in CFA and T cells using TRIzol reagent (Life Technologies). cDNA was synthesized with received 200 ng of Pertussis toxin intraperitoneally on days 0 and 2. Mice iScript Reverse Transcription Supermix for RT-qPCR (Bio-Rad), followed by were monitored daily for disease progression. At the end point, the brain qPCR using SYBR Green PCR Master Mix (Applied Biosystems). Quantitative and spinal cord were harvested for histology and immune cell profiling. For PCR was performed on an Applied Biosystems ViiA 7 Real-Time PCR System passive EAE, CD4 T cells from Rosa-M2rtTAxTRE-RVBx2D2 mice were acti- with gene specific primers listed in SI Appendix, Table S1. vated under Th17 condition for 3 d, then restimulated overnight in the presence of IL-18 (20 ng/mL; Fisher Scientific). Two to 3 million T cells were Retroviral Transduction. HEK 293T cells were transfected via FuGENE6 reagent adoptively transferred into WT recipient mice, which were given normal (Promega), which contained 0.8 μg of pCL-Eco retroviral packaging plasmid water or Doxycycline water to induce REV-ERBα expression. EAE disease and 1.2 μg of expression plasmid. pCL-Eco was a gift from Inder Verma (Salk progression was monitored as in the active EAE model. Institute, La Jolla, CA) (38). Viral supernatant was harvested 48 and 72 h + after transfection. CD4 T cells were cultured in Th17 polarizing condition ACKNOWLEDGMENTS. We thank A. Cheng, Y. Zhang, and C. Gordon for and retroviral transduction was performed 24 and 48 h after activation by mouse colony management and Y. Dai, M. Ku, S. Heinz, and C. Benner for incubating cells with viral supernatant in the presence of polybrene (4 μg/mL; assistance in RNA-seq experiments. C.C. is supported by the H.A. and Mary Millipore) and centrifuged at 2,500 rpm for 90 min at 32 °C. K. Chapman Charitable Trust. C.-S.L. is supported by the Albert G. and Olive H. Schlink Foundation. S.P.B. is supported by NIH Grants DK096828 and T32 + ChIP. Naive CD4 T cells were activated and polarized in Th17 condition for GM007198. R.M.E. is an Investigator of the Howard Hughes Medical Institute 3 d for ChIP experiments as described previously (14). Mouse IgG control at the Salk Institute and March of Dimes Chair in Molecular and De- antibody was purchased from Santa Cruz Biotechnology. RORγt ChIP was velopmental Biology and is supported by NIH Grants HL088093 and HL105278, Leona M. and Harry B. Helmsley Charitable Trust Grant 2017PG- performed with a combination of antibodies from BioLegend and Santa MED001, Ipsen/Biomeasure, and the Fondation Leducq. Y.Z. is supported by Cruz Biotechnologies. REV-ERBα antibody was generated as previously de- the NOMIS Foundation, the Rita Allen Foundation, National Multiple Sclerosis scribed (14). NCoR1 antibody was purchased from Cell Signaling. Primers Society Grant RG4978-A-2, and NIH Grants AI107027 and OD023689. This work spanning the regulatory regions of Il17a, Cry1, and Gmpr are described in SI was also supported by National Cancer Institute-funded Salk Institute Cancer Appendix, Table S2. Center core facilities Grant CA014195. Research reported in this publication was also supported by the National Institute of Environmental Health Sciences ChIP-Seq and Data Analysis. ChIP-seq libraries were constructed and sequenced of the NIH under Award P42ES010337. The content is solely the responsibility as described previously (14). Reads were aligned against the mouse mm9 of the authors and does not necessarily represent the official views of the NIH.

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